Pilot-scale fermentation, purification, and characterization of recombinant human Oncostatin M in Pichia pastoris.
Oncostatin M (OSM) is a multifunctional cellular regulator that belongs to the IL-6 subfamily and can act on a wide variety of cells, which has potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. In order to achieve the higher level yield of recombinant human Oncostatin M (rhOSM), we determined the optimal pH condition of rhOSM expressed in the methylotrophic yeast Pichia pastoris X-33 and carried out the fermentation culture of rhOSM in 80 L fermentor in a fed-batch mode. SDS-PAGE and Western blotting assays demonstrated that rhOSM was successfully expressed and secreted into the culture medium with an apparent molecular weight of 28 kDa. N-terminals were correctly processed through amino-terminal sequencing. The maximum yield of rhOSM was 280 mg/L. rhOSM was purified by phenyl Sepharose hydrophobic interaction chromatography and SP Sepharose Fast Flow cation exchange chromatography, which resulted in a final yield of purified rhOSM of 6.94 g with a recovery of 62% and a purity of 95%. The purified rhOSM had a specific growth inhibition activity of 6.26 x 10(4)RU/microg, which was commensurate with typical values (6.2 x 10(4)RU/microg) obtained with standard hOSM.
Kong
Ning
N
Department of Biological Engineering, College of Pharmacy, Jilin University, 1163 Xin Min Street, Changchun 130021, PR China.
Mu
Xupeng
X
Han
Hongzhi
H
Yan
Weiqun
W
D019149:Bioreactors; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003001:Cloning, Molecular; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D053683:Oncostatin M / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D010843:Pichia / Q000235:genetics; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology
D011994:Recombinant Proteins; D053683:Oncostatin M